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Abstract

Oxidative stress causes profound alterations of various biological structures, including cellular membranes, lipids, proteins and nucleic acids, and it is involved in numerous malignancies. Reduced glutathione (GSH) is considered to be one of the most important scavengers of reactive oxygen species (ROS), and its ratio with oxidised glutathione (GSSG) may be used as a marker of oxidative stress. The main aim of this study was to determine GSH:GSSG ratio in the blood serum of paediatric cancer patients to use this ratio as a potential marker of oxidative stress. The whole procedure was optimised and the recoveries for both substances were greater than 80% under the optimised conditions. We analysed a group of paediatric patients (n=116) with various types of cancer, including neuroblastoma, anaplastic ependymoma, germ cell tumour, genital tract tumour, lymphadenopathy, rhabdomyosarcoma, nephroblastoma, Ewing's sarcoma, osteosarcoma, Hodgkin's lymphoma, medulloblastoma and retinoblastoma. We simultaneously determined the levels of reduced and oxidised glutathione, and thus, its ratio in the blood serum of the patients. The highest ratio was observed in retinoblastoma patients and the lowest in anaplastic ependymoma. We were able to distinguish between the diagnoses based on the results of the obtained GSH:GSSG ratio.

Introduction

Reduced glutathione (GSH), a ubiquitous tripeptide
thiol, is a vital intracellular and extracellular protective
antioxidant, which plays a number of key and/or crucial roles in
the control of signalling processes, detoxifying certain
xenobiotics and heavy metals, as well as other functions. It is a
tripeptide composed of cysteine, glutamic acid and glycine.
Intracellular and whole blood concentrations of GSH are in the
milimolar range, while the plasma concentration is in the
micromolar range and accounts for approximately 0.4% of total blood
GSH (1–5). The GSH synthesis and metabolism
pathway is shown in Fig. 1. GSH is
synthesised in the cell by γ-glutamylcysteine synthetase (γ-GCS)
and glutathione synthetase (6). The
γ-GCS-catalysed formation of γ-glutamylcysteine is the first and
rate-limiting step in de novo GSH synthesis and is
feedback-inhibited by GSH, a mechanism that is central to the
regulation of cellular GSH concentrations (7). Thus, cysteine is a rate-limiting
substrate for de novo GSH synthesis (8).

Within cells, total GSH exists free and bound to
proteins. Since the enzyme glutathione reductase, which reverts
free glutathione from its oxidised form (GSSG) is constitutively
active and inducible upon oxidative stress, free glutathione exists
almost exclusively in its reduced form. The ratio of reduced to
oxidised glutathione within cells is often used as a marker of
cellular toxicity (9–12). Under normal conditions, the GSH
redox couple is well-known to be present in mammalian cells in the
concentration range of 1–10 mM. In a resting cell, the molar
GSH:GSSG ratio exceeds 100:1, while in various models of oxidative
stress, this ratio has been demonstrated to decrease to values of
10:1 and even 1:1 (13).

Oxidative stress is manifested by the excessive
production of reactive oxygen species (ROS) in the face of
insufficient or defective antioxidant defence systems. Oxidative
stress causes profound alterations of various biological
structures, including cellular membranes, lipids, proteins and
nucleic acids. Oxidative stress is considered to be involved in
ageing (14–20) and in various diseases, including
diabetes mellitus (21–23), atherosclerosis (24,25),
rheumatoid arthritis (26–29), Alzheimer’s disease (30–32),
Parkinson’s disease (33–35) and cancer (36–44).
There is an increasingly growing interest in identifying biomarkers
for diseases, in which oxidative stress is involved (45).

For many years, GSH has been measured by several
analytical methods. In particular, high performance liquid
chromatography (HPLC) with various detection techniques including
ultraviolet (UV) absorbance and fluorescence detection, mass
spectrometry and/or electrochemical detection (ED) are commonly
used for determination of GSH and GSSG concentrations (46–49).
Each method has its advantages and limitations and may serve a
particular need in analysis (50).
ED is an attractive alternative method for electroactive species
detection, due to its inherent advantages of simplicity, ease of
miniaturisation, high sensitivity and relatively low cost. The aim
of this study was to determine the GSH:GSSG ratio in the blood
serum of paediatric cancer patients to use this ratio as a
potential marker of oxidative stress. For determination of the
GSH:GSSG ratio, HPLC-ED was optimised and used.

HPLC-ED analysis

The HPLC-ED system consists of two chromatographic
pumps (Model 582; ESA, Inc., Chelmsford, MA, USA; working range
0.001–9.999 ml/min), a chromatographic column with reverse phase
Zorbax eclipse AAA C18 (Agilent Technologies, Inc., Santa Clara,
CA, USA; 150x4.6 mm; 3.5-μm particles) and a twelve-channel
CoulArray electrochemical detector (Model 5600A; ESA, Inc.). The
detector consists of three flow analytical chambers (Model 6210;
ESA, Inc.). Each chamber contains four analytical cells and one
analytical cell contains two referent (hydrogen-palladium), as well
as two counters and porous graphite working electrodes. The ED is
situated in the thermostated control module. A 20 μl sample
was injected using an autosampler (Model 542; ESA, Inc.), which has
thermostated space for the column. The column was termostated at
35°C. Other conditions were optimised and are described later.

Determination of recovery in real
samples

Recovery of GSH and GSSG were evaluated with
homogenates spiked with standards according to Causon (50). Prior to extraction, 100 μl
GSH and GSSG was added to the blood serum homogenate. Homogenates
were blindly assayed and the concentration of GSH and GSSG was
derived from the calibration curves. The spiking of GSH and GSSG
was determined as a standard measured in the absence of real
sample. Accuracy was evaluated by comparing the estimated
concentration with the known concentrations of both thiols.

Descriptive statistics

Data were processed using Microsoft Excel (USA).
Results are expressed as mean ± standard deviation (SD) unless
otherwise stated. The detection limits [3 signal/noise (S/N)] were
calculated according to Long and Winefordner (51), while N was
expressed as the SD of noise determined in the signal domain unless
otherwise noted.

Results

Optimisation of HPLC-ED method

Primarily, it was necessary to optimise the
separation and subsequent ED in order to achieve the required
accuracy and sensitivity for the determination of GSH and GSSG in
real blood serum samples. Therefore, we focused on studying the
influence of flow rate, concentration of components of the mobile
phase, elution and applied potential of the working electrodes on
GSH and GSSG signals.

Flow rate

The mobile phase flow rate is an important parameter
influencing the electrochemical response of the detector. When
using a chromatographic column Zorbax Eclipse AAA, optimum mobile
phase flow rate was 1 ml/min at pressures of 130 bars.
Additionally, we identified that if the flow >1 ml/min, the
responses of GSH and GSSG decreased by >10%. This is probably
caused by reducing the time-concentration of the analyte on the
electrode surface. Even with a lower flow rate, a decreased signal
occurred compared with the maximum, but the total peak area
remained the same with a tolerance of 7%. Although a lower flow
rate may not be significantly affected by resolution, it may extend
the period of separation, which is critical for analysing a large
number of clinical samples. Therefore, we decided to use 1 ml/min
as the optimum flow rate of the mobile phase.

Influence of methanol on ED

Achieving an optimal resolution is crucial for
simultaneous separation of analytes. In order to separate all
determined substances in the system with reversed-phase, a gradient
with the increasing content of organic solvent is required. Since
the electrochemical determination of substances contained in the
sample requires the presence of an electrolyte, we examined the
effect of the organic solvent (methanol) on the electrochemical
response of analytes. We identified that 15% content of methanol in
the mobile phase, which is the polar component of the mobile phase
composed also from 80 mM TFA, lead to more than 50% decrease in GSH
signal. A marked decline of GSSG signal was also observed. The best
ratio of 80 mM TFA and methanol in the mobile phase was 99:1
(v/v).

Optimisation of gradient

If GSH and GSSG were separated by isocratic elution
where the ratio of TFA and methanol was 99:1 (v/v), it would be the
most sensitive, but the retention times of the separated substances
would be too high. A significant tailing of peaks was observed
during the elution of compounds with higher retention under these
conditions. Therefore, we optimised the increasing content of
methanol with respect to the sensitivity of the analysis. Based on
the optimisation steps, the mobile phase, which consisted of (A) 80
mM TFA and (B) 100% methanol, was used for separation and detection
of GSH and GSSG. Compounds were eluted by following an increasing
linear gradient: 0–1 min (3% B), 1–2 min (10% B), 2–5 min (30% B)
and 15–16 min (98% B). Flow rate of the mobile phase was 1 ml/min,
and the time of one analysis inducing column regeneration was 20
min.

ED

Sensitivity of the electrochemical detector may be
more influenced by factors including the type of electrolyte in the
mobile phase, concentration, pH and, in particular, applied
potential. TFA was used as an ion-pair reagent, which provides the
best separation conditions in the parameters mentioned above, and
at a concentration of 80 mM it is also an extremely suitable
electrolyte for the detection of thiols. We further studied the
effect of the applied potential on the working electrode set
separately for GSH and GSSG, which were designed for hydrodynamic
voltammogram (HDV). Tested potentials were 100, 200, 300, 400, 500,
600, 700, 800, 900 and 1,000 mV. The responses detected at 100 mV
were negligible; however, when the potential reached 300 mV,
detectable signals for GSH and GSSG were observed. While the GSH
signal markedly increased from 600 mV, the GSSG signal markedly
increased from 700 mV. This is probably due to the requirement for
greater power for partial dissociation of the -S-S- group on the
surface of the working electrode, in comparison to the relatively
easily accessible -SH moiety of GSH. We observed the highest
signals for both compounds when a potential of 900–1,000 mV was
applied, which is evident from the HDVs showed in Fig. 2A. Based on the HDV results we were
able to evaluate that the best glutathione detection was achieved
when a potential of 900 mV was applied to the working
electrodes.

Calibration parameters

After identifying the optimal separation and
detection conditions, the calibration curves for GSH and GSSG were
measured within the concentration range of 0.2–100 μM.
Overlay of HPL chromatograms is shown in Fig. 2B, and the calibration curves are
shown in Fig. 2C. The obtained
dependences were strictly linear with R2=0.9997 for GSH
and R2=0.9936 for GSSG. Detection limits (3 S/N) were
estimated with nanomolar subunits for both substances of
interest.

Sample pretreatment for GSH:GSSG ratio
determination

Prior to chromatographic analysis, precipitation of
proteins with TFA to avoid excessive clogging of filters and
precolumns, which protect the separation column from
contaminations, was required. The proteins may interfere with
detected substances and the obtained chromatograms may be extremely
difficult to analyse. The denatured sample was than centrifuged and
the resulting supernatant was directly injected to the
chromatographic column. To ensure the lowest possible loss of
target compounds during sample preparation it was necessary to
examine several factors of a sample treatment, which could affect
the overall recovery of GSH and GSSG.

Stability of GSH

Given that the formation of complexes may be faster
under certain conditions (pH and ionic strength), we decided to
investigate the possibility of GSH complex formation in the
solution used for isolation. The formation of the complex was
determined via a decrease in the GSH peak. Primarily, we examined
the effect of molar concentrations of phosphate buffer (0.1, 0.5,
1, 5, 10 and 20 mM; pH 7.5) on the GSH (50 μg/ml) signal.
These samples were left following preparation at room temperature,
and were analysed by HPLC at time 0, 130 (2.2 h) and 260 min (4.3
h). Based on the results obtained, higher concentrations of buffer
caused a decreasing GSH signal, i.e. concentration; thus, 20 mM
phosphate buffer caused the highest decrease of glutathione
concentration. It is clear that the greatest stability of GSH was
observed in samples prepared in the presence of low concentrations
of phosphate buffer.

Specifically, the lowest loss of glutathione
occurred at the applied concentrations of 0.1–1 mM (Fig. 3A). These results clearly
demonstrated that lower concentrations of phosphate buffer
contribute to the stability of the sample. Therefore, for further
experiments we used 1 mM phosphate buffer (pH 7.5).

Influence of various chemicals on
GSH:GSSG ratio

To determine the extent of oxidative stress by
glutathione it is necessary to know the ratio of GSH:GSSG.
Therefore, we were aimed to determine whether TFA, which is
normally added to the sample due to deproteination, could have an
effect on GSSG level. We also studied the effect of adding the
reducing agent tris(2-carboxylethyl)phosphine (TCEP), which may
markedly influence the ratio of GSH:GSSG. Studies on TFA and TCEP
were conducted in buffer and blood sera, and all variants were
prepared with the same concentration of 50 μg/ml GSH and 5
μg/ml GSSG. Samples were prepared in the presence of (i) 1
mM phosphate buffer (pH 7.5), (ii) 1 mM phosphate buffer (pH 7.5)
with 5% TFA (v/v), and (iii) 1 mM phosphate buffer (pH 7.5) with 1
mM TCEP. To be able to assess the influence of the matrix, samples
of blood serum were prepared in the same way. All samples were
vortexed for 1 min and immediately analysed by HPLC following
preparation. The GSH:GSSG ratio was determined, where the ratio of
10 was taken as a control. In the case of using 5% TFA, ±7% change
from control was determined in variants of buffer and serum
(Fig. 3B). The results reveal that
TFA did not affect the ratio of GSH:GSSG. However, following the
addition of TCEP, there was a significant increase in the ratio to
38 and 48 in the buffer and blood serum, respectively. TCEP reduced
the majority of GSSG to GSH, which was the reason for the
significant increase of the GSH:GSSG ratio. In the case of blood
serum, the ratio was even higher compared with buffer. This
phenomenon may be explained by the involvement of the biological
matrix in a non-specific reaction of the complexes or the presence
of certain concentrations of glutathione bound to the matrix
constituents. These results clearly indicate that TCEP reduces GSSG
back to GSH, which could be used to determine the total amount of
glutathione.

Recovery of pretreatment

Recovery estimation for sample preparation and
analysis for a sample of blood serum using an optimised separation
method was conducted by adding 10 μg/ ml GSH and 10
μg/ml GSSG prior to precipitation with 5% TFA and subsequent
centrifugation. A sample with a GSH:GSSG ratio of 2.8 was used for
determining recovery. The resulting recoveries are indicated in
Table I. A recovery estimation of
83 and 89% for GSH and GSSG, respectively, clearly follows from the
results previously obtained. GSH recovery can be associated with
the imperfect protection of free-SH groups of glutathione, which
can interact with the remains of biological matrices, and thus
reduce the total concentration of free GSH during the preparation
of the samples.

Table I.

Recovery of GSH and GSSG for blood
serum sample analysis (n=5).

Table I.

Recovery of GSH and GSSG for blood
serum sample analysis (n=5).

Substance of
interest

Homogenate
(μg/ml)

Spiking
(μg/ml)

Homogenate +
spiking (μg/ml)

Recovery (%)

GSH

54±6

50±5

86±10

83

GSSG

25±4

10±2

31±3

89

[i] GSH, reduced
gluthione; GSSG, oxidised gluthione.

Determination of GSH:GSSG ratio in
paediatric patients

The antioxidant function of GSH is primarily due to
its involvement in enzymatic pathways that cells have developed
against ROS. The most important pathway involves glutathione
peroxidase (GPx) and glutathione reductase (GR). GPx catalyses the
reduction of hydrogen peroxide, which is produced by superoxide
dismutase (SOD) through the dismutation of superoxide anions or
organic hydroperoxides. GSH and GSH-dependent enzymes act in
cooperation to scavenge ROS and/or neutralise their toxic oxidising
effect. These systems act at the same time and in cooperation to
protect the human body from ROS. Under oxidative stress conditions,
GSH is oxidised to GSSG; thus, the GSH:GSSG ratio is altered.

Discussion

The GSH:GSSG ratio may be used as a marker of
oxidative stress, which arises due to various malignancies. Using
the optimised method, we were able to analyse real samples of
paediatric patients (Fig. 4A). GSH
and GSSG concentrations identified in each sample were recalculated
to recovery, and based on these values, the GSH:GSSG ratios were
given. The lowest number of patients in a group (n=3) were
diagnosed with lymphadenopathy and the highest number (n=27) were
diagnosed with neuroblastoma. Average values of GSH:GSSG ratio are
demonstrated in Fig. 4B. The
results reveal that the lowest redox status, which is given by the
GSH:GSSG ratio of 1.4, was identified in patients diagnosed with
ependymoma anaplastic, and the second lowest ratio of 1.5 was
identified in patients diagnosed with genital tract tumour. The
average values of both groups of patients also had a large relative
standard deviation (RSD) of 50.3 and 41.5%, respectively. The
lowest RSDs were identified in lymphadenopathy and rhabdomyosarcoma
patients with a higher GSH:GSSG ratio of 4.0 and 3.5, where RSDs
were 18.4 and 22.1, respectively. Additionally, the lowest
oxidative damage, expressed as a GSH:GSSG ratio of 5.2, was
revealed in retinoblastoma patients.

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